半胱氨酸天冬氨酸蛋白酶-3（Caspase-3）的激活会导致严重的神经元损伤和学习记忆障碍。前一项目的研究表明：脑缺血可促使血红素加氧酶-1（HO-1）与MLK3-MKK7-JNK3信号模块结合，从而激活Caspase-3。可是，HO-1是与该模块的支架蛋白JIP1直接作用，还是与MLK3、MKK7或JNK3作用，及具体作用位点，尚不清楚。该位点若能探明，则可能成为新的药物靶点。 本项目拟采用哺乳动物细胞双杂交，从JIP1、MLK3、MKK7和JNK3中筛选与HO-1直接作用的"目的蛋白"，以GST pull-down、免疫沉淀和激光共聚焦验证之；分别构建HO-1和"目的蛋白"的位点突变体，再以上述技术筛选并验证两者之间的作用位点；突变该位点，探索该位点在脑缺血后JNK3 /Caspase-3通路的调控、神经元凋亡和学习记忆中的作用及机制。本项目可为该病的治疗提供新的药物靶点和实验依据。

The activation of cysteinyl aspartate-specific protease-3 (Caspase-3) induced by ischemia and reperfusion could cause severe neuron injury and disorder of learning and memory. During the research of the previous project, it was indicated that brain ischemia could facilitate heme oxygenase -1 (HO-1) to combine with MLK3-MKK7-JNK3 signal module and increase the activation of Caspase-3. However, which protein of JIP1, MLK3, MKK7 or JNK3 can combine with HO-1, and where are interacting sites between them, are still unclear. These interacting sites may become new drug targets if they are found in the future. In this project, mammalian two-hybrid system will be used to sieve the "interest protein" which can combine with HO-1 directly from JIP1, MLK3, MKK7 and JNK3; GST pull-down, immunoprecipitation and confocal laser scanning microscope (CLSM) will be used to validate the above experimental results; The sites mutagenesis of HO-1 and "interest protein" will be prepared to sieve and validate the interacting sites between HO-1 and "interest protein" using the above technologies. These interacting sites will be mutated to explore the effects and relative mechanisms of them on the regulating JNK3 /Caspase-3 signaling pathway, neuron apoptosis, learning and memory after brain ischemia. This project will provide new drug targets and experimental evidences for the therapy of the ischemic stroke.